Strategies to combat HIV-1 require structural knowledge of how antibodies recognize HIV envelope proteins and how they are used by the immune system to eliminate viruses and virally-infected cells. Until recently, only a small number of broadly neutralizing antibodies (bNAbs) against HIV-1 had been characterized and the immunological basis for their breadth and potency remains poorly understood. One important obstacle to a comprehensive understanding of how antibodies neutralize HIV was the limited availability of monoclonal bNAbs. This issue was resolved by new methods for HIV antibody isolation from B cells of patients who produce high titers of broad neutralizing activity. Several of the antibodies recently isolated by Dr. Michel Nussenzweig's laboratory, including NIH45-46, 3BNC117 and 3BNC60, show superior potency and breadth to VRC01, previously the best bNAb. We recently solved the structure of NIH45-46 bound to gp120, and used structure-based design to increase its potency by at least 10-fold, making NIH45-46G54W the most potent anti- HIV bNAb currently available. We will collaborate with Dr. Nussenzweig to determine the structural correlates of broad and potent neutralization by the variable Fabs of new bNAbs as they are isolated from new donors. Basic knowledge of bNAb efficacy against HIV will facilitate optimization of the breadth and potency of natural human monoclonal antibodies for passive delivery by immunization or gene therapy and provide to the underlying framework needed to develop active immunization strategies that will elicit bNAbs. The following specific aims will be pursued: 1) Solve crystal structures of bNAb Fabs and bNAb Fab-gp120 complexes to define the essential features of broad and potent HIV neutralization. 2) Produce natural antibody variants with enhanced activity in neutralization. PUBLIC HEALTH RELEVANCE: HIV/AIDS remains one of the most important current threats to global public health. At least 60 million people have been infected with HIV, of which almost half have died. Although anti-retroviral drugs have been effective in the developed world, a vaccine and/or new methods to prevent infections are urgently needed in the developing world. We will systematically analyze new anti-HIV antibodies with goals of understanding their protective mechanisms and improving antibodies for passive delivery prevention strategies.